Various dry analytical assay elements are commercially available, such as Kodak's Ektachem Clinical Chemistry slides, and certain known dry multilayer immunoassay elements, include a layer comprising a leuco dye. In the course of an assay for a specified analyte in a sample, the leuco dye, in the presence of hydrogen peroxide and a material having peroxidase activity, is oxidized to a colored form. As is well known the reflection density of the color is proportional to the concentration of analyte in the sample. The reflection density can be measured using a reflectometer. See U.S. Pat. Nos. 4,670,385; 4,089,747; 5,024,935; 4,089,747 and 4,258,001 for further details and references to other literature describing this art.
Typical leuco dyes used for this purpose are highly aromatic leuco dyes that cannot be dissolved and deposited or coated as an aqueous solution. Such leuco dyes are the diaryl- and triarylmethanes of U.S. Pat. No. 4,670,385; and the diaryl- and triarylimidazole dyes of U.S. Pat. No. 4,089,747 and U.S. Pat. No. 5,024,935. Such dyes have been coated as dispersions by dissolving the dye in an organic solvent (methanol or dimethyl sulfoxide) and reprecipitating in an aqueous polymer solution to produce a coating composition of the dye in a polymer solution; or dissolving the dye in a "coupler solvent" (diethyl lauramide) and redispersing the coupler solvent solution in an aqueous solution.
Another process for making such dye coating compositions is to dissolve the dye in a good solvent for the dye (dimethyl sulfoxide), and reprecipitate the dye in an aqueous coating composition. This requires very careful control of the precipitation process in order to optimize the particle size of the dye in the coating composition. This is difficult to do because the process is sensitive to stir rate and rate of addition of the dye solution, thus large particles are formed, and consequently more dye than necessary must be employed for adequate color formation in an assay. Further, the process is unpredictably irreproducible, because, in addition to the above, the dye tends to coagulate, and the coating composition has limited shelf life. It is also desirable to avoid the use of DMSO.
There is a great need for immunoassay elements comprising stable leuco dye coating compositions form without use of organic solvents, that use of up to half as much dye, are reproducible, and do not require stringent process control operations.